Abstract Metabolic rewiring supports glioblastoma (GB) progression, yet the contribution of fatty acid oxidation (FAO) to GB metabolic plasticity remains poorly defined. GB tumors display elevated expression of FAO-related genes, including carnitine palmitoyltransferase 1A (CPT1A), suggesting a potential reliance on this pathway. Here, we evaluated the functional relevance of FAO in U251 GB cells using etomoxir (ETO), a CPT1A inhibitor. ETO markedly reduced cell viability in monolayers (5-day exposure) and 3D spheroids (9-day exposure; IC50=118 µM) and rapidly disrupted spheroid architecture within 48 hours. Short-term treatment with 200 µM ETO did not alter cell size or granularity, but FAO inhibition induced a clear metabolic shift characterized by increased glucose consumption, elevated lactate release, and enhanced extracellular acidification, consistent with compensatory glycolytic upregulation. To test whether this adaptive response creates a metabolic vulnerability, we inhibited glycolysis with 2-deoxyglucose (2DG). Combined ETO+2DG treatment significantly potentiated cytotoxicity compared with either agent alone after 72 hours. These findings indicate that FAO serves as a relevant energy source in GB cells and that its inhibition triggers increased glycolytic flux as a compensatory mechanism. Together, our data reveal a targetable FAO-glycolysis crosstalk in GB and support the therapeutic potential of dual metabolic pathway inhibition to exploit GB metabolic flexibility. Citation Format: Lola Martinez Ibarguren, Fiorella Orsini Zanetti, Sofia Paz Osorio Rencoret, Maria Florencia Arbe, Marina Perona, Gabriela Salamone, Gerardo Martin Oresti, Pablo J Sáez, Catalina Lodillinsky, Marcela Solange Villaverde. Metabolic crosstalk between fatty acid oxidation and glycolysis underlies glioblastoma viability abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3283.
Ibarguren et al. (Fri,) studied this question.